Therapeutic method

ABSTRACT

The present invention relates to new therapeutic use of amylin as agent which stimulates chondrocyte proliferation and which therefore have utility in the treatment of cartilage disorders and/or cartilage mediated bone growth.

This application is a 371 of PCT/NZ98/00145 filed 25 Sep. 1998, whichclaims foreign priority benefit of the filing date under 35 U.S.C. 119of New Zealand patent No. 328853, filed 29 Sep. 1998.

This invention is directed to new therapeutic uses which involve thestimulation of chondrocyte proliferation. More particularly, it isdirected to the use of amylin and adrenomedullin as agents whichstimulate chondrocyte proliferation and which therefore have utility inthe treatment of cartilage disorders and/or cartilage mediated bonegrowth.

BACKGROUND

Amylin is a 37-amino acid peptide cosecreted with insulin from the betacells of the pancreatic islets. It was first reported by Cooper et al inProceedings of the National Academy of Sciences, USA 84, 8628 (1987) andis the subject of European Patent 289287. Amylin has the followingpeptide sequence:

(SEQ ID NO:1) Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln- 1                5                 10Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-11              15                   20Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-21               25                  30 Asn-Val-Gly-Ser-Asn-Thr-Tyr31              35

The native molecule contains a disulphide bridge between the cysteineresidues shown at positions 2 and 7 in the primary structure, isamidated at its COOH-terminus, and is formed as a propeptide.

European Patent 289287 reports a number of biological effects includingenhancement of hepatic glucose output, increased production of lactatefrom skeletal muscle and reduced action of insulin in skeletal muscle.

Amylin is also reported in European Patent 408284 as having value fortreatment of bone disorders and calcium imbalance. The patentspecification attributes the activity of amylin to an inhibition ofosteoclast motility. It is also resorted in WO 96/02269 as stimulatingbone growth through stimulating osteoblas; proliferation.

Adrenomedullin is a 52-amino acid peptide first described in 1993 byKitamura et at (Kitamura, K., et al. Adrenomedullin, a novel hypotensivepeptide isolated from human pheochromocytoma. Biochem. Biophys. Res.Commun. 192:553-560 (1993)). It is present in normal adrenal/medulla andin many other tissues including the atria, ventricles, endothelialcells, lungs, brain, kidneys and bone.

Adrenomedullin shows approximately 20% sequence identity with amylin andcan therefore be termed a related peptide (Muff, R., et al. Calcitonin,calcitonin gene-related peptide, adrenomedullin and amylin: homologouspeptides, separate receptors and overlapping biological actions. Eur. J.Endocrinol. 133:17-20 (1995)). Both peptides have an NH₂ terminal ringcreated by a disulphide bond and are amidated at their COOH terminals.

Like amylin, adrenomedullin is also reported to have a range ofactivities. It is a potent vasodilator. It also has value in thetreatment of bone disorders. This is primarily through an ability tostimulate osteoblast activity and proliferation in vitro and in vivo(Cornish, J., at al Adrenomedullin is a potent stimulator ofosteoblastic activity in vitro and in vivo. Am. J. Physiol (EndocrinolMetab) 36:E1113-E1120, (1997)).

However, to date, there has been no report of either of the peptidesamylin or adrenomedullin, as having any effect on chondrocytes. It isthe applicants finding that both of these peptides are effective in thestimulation of chondrocyte proliferation and therefore on the growth ofboth cartilage and lineal bone. This effect is believed to be mediatedthrough a single receptor on chrondrocytes which underlies theapplicant's invention.

SUMMARY OF THE INVENTION

The invention has a number of aspects. In a first aspect, the inventionprovides a method of treating a patient to stimulate chondrocyteproliferation in vivo which comprises the step of increasing the activeconcentration of amylin within said patient.

Another aspect provides a method of treating a patient to stimulatechondrocyte proliferation in vivo which comprises the step ofadministering to said patient amylin or an analog thereof in an amounteffective to stimulate chondrocyte proliferation.

In another embodiment, the invention provides a method of treating apatient to stimulate chondrocyte proliferation in vivo which comprisesthe step of increasing the active concentration of adrenomedullin withinsaid patient.

In a further embodiment, the invention provides a method of treating apatient to stimulate chondrocyte proliferation in vivo which comprisesthe step of administering to said patient adrenomedullin or an analogthereof in an amount effective to stimule chondrocyte proliferation.

In still a further aspect, the invention provides a method of treating apatient to stimulate chondrocyte proliferation in vivo which comprisesthe step of activating the receptor localised on chondrocytes of saidpatient to which amylin and/or adrenomedullin bind.

Most preferably, the method involves activation of the adrenomedullinreceptor.

Conveniently, in each of the above methods the stimulation ofchondrocyte proliferation is part of a method of treating a patient tostimulate cartilage growth and/or repair or to stimulate bone growth.

The invention also provides a method of stimulating chondrocyteproliferation in vitro which comprises administering an amount ofamylin, adrenomedullin or an analog of either amylin or adrenomedullinto said chondrocytes which is effective in inducing chondrocyteproliferation.

Other aspects include:

-   -   the use of amylin or an analog thereof in the preparation of a        medicament for effecting chondrocyte proliferation;    -   the use of adrenomedullin or an analog thereof in the        preparation of a medicament for effecting chondrocyte        proliferation;    -   the use of a ligand which binds to and activates the receptor to        which amylin and/or adrenomedullin binds (preferably the        adrenomedullin receptor) in the preparation of a medicament for        effecting chondrocyte proliferation;    -   the use of an amylin agonist in the preparation of a medicament        for effecting chondrocyte proliferation;    -   the use of an adrenomedullin agonist in the preparation of a        medicament for effecting chondrocyte proliferation;    -   the use of amylin-(1-8) (SEQ ID NO:2) in the preparation of a        medicament for effecting chondrocyte proliferation; and    -   the use of adrenomedullin-(27-52) (SEQ ID NO:3) in the        preparation of a medicament for effecting chondrocyte        proliferation.

DESCRIPTION OF THE DRAWINGS

The present invention is broadly as defined above. However, it will beappreciated by those persons skilled in the art that it is not limitedthereto and that it also includes embodiments which are moreparticularly described below and illustrated by the experimental datapresented. This data includes the information shown in the accompanyingdrawings in which:

FIG. 1 shows the effects of daily systemic administration of amylin for4 weeks on growth plate width in the tibiae of normal adult male mice.n=20 in each group. ^(★), significantly different from control,P=0.0002;

FIG. 2 shows the effects of daily systemic administration of amylin for4 weeks on bone length in the tibiae of normal adult male mice. n=20 ineach group. ^(★), significantly different from control, P=0.004;

FIG. 3 shows the effect of the amylin fragment (amylin (1-8)) onepiphyseal growth plate width; and

FIG. 4 shows the effect of the adrenomedullin fragment (adm 27-52) onepiphyseal growth plate width.

DESCRIPTION OF THE INVENTION

As broadly defined above, the present invention relates primarily tomethods for stimulating chondrocyte proliferation. The inventiontherefore has utility in any application where stimulation ofchondrocyte proliferation or growth is viewed as desirable, includingfor example cartilage growth and bone growth.

The applicants have found that chondrocyte proliferation is able to beeffected using a number of related approaches. A first such approach isthrough a focus upon amylin. The applicants have found that increasingthe effective concentration of amylin within a patient able to interactwith the patients chondrocytes has the effect of stimulating chondrocyteproliferation.

Amylin for use in accordance with this approach can be obtained from anyconvenient commercial source (such as Bachem Calif., Torrence, Calif.,USA). Alternatively, amylin can be synthesised, using the procedure asdescribed by way of example in EP 408284.

The amylin used can be homologous or heterologous to the patient to betreated. For example, amylin from humans and other mammals eg. rat,monkey, dog, cat, mouse, guinea pig, hamster, degus, rabbit and hare canbe used. The structure of these various peptides is reported inEndocrine Reviews 1994, 15(2) 163 by Garth J S Cooper which isincorporated herein by reference.

Most conveniently, the effective concentration of amylin will beincreased through direct administration using either amylin itself or anamylin pro-drug (a form which is cleaved within the body to releaseamylin). It is however not the applicants intention to excludeincreasing amylin concentration through administration of either amylinagonists (substances which effect a direct increase in the production oractivity of amylin within the body, or inhibitors of amylin antagonists(substances which bind amylin or otherwise prevent or reduce the actionof amylin within the body, These latter compounds exert an indirecteffect on effective amylin concentrations through the removal of aninhibitory mechanism.

Another possibility is administration of a replicable vehicle encodingamylin to the patient. Such a vehicle (which may be a modified cell lineor virus which expresses amylin within the patient) could haveapplication in increasing the concentration of amylin within the patientfor a prolonged period.

It is also contemplated that amylin analogs can be employed in thisinvention. As used herein “analog” means a protein which is a variant ofanother protein through insertion, deletion or substitution of one ormore amino acids but which retains at least substantial functionalequivalency.

A protein is a functional equivalent of another protein for a specificfunction if the equivalent protein is immunologically cross-reactivewith, and has at least substantially the same function as, the originalprotein. The equivalent can be, for example, a fragment of the protein,a fusion of the protein with another protein or carrier, or a fusion ofa fragment with additional amino acids. For example, it is possible tosubstitute amino acid in a sequence with equivalent amino acids usingconventional techniques. Groups of amino acids normally held to beequivalent are:

-   -   (a) Ala, Ser, Thr, Pro, Gly;    -   (b) Asn, Asp, Glu, Gln;    -   (c) His, Arg, Lys;    -   (d) Met, Leu, Ile, Val; and    -   (e) Phe, Tyr, Trp.

In the case of amylin, the preferred analogs are fragments of theprotein. In particular, amylin (1-8) can be used (ie. a fragmentconsisting of amino acids 1 to 8 of the amylin sequence).

Functional equivalency of analogs can also be readily screened for byreference to the ability of the analog to both bind to and activate theappropriate receptor.

In addition to the above approach, which focuses upon amylin and itsanalogs, the invention provides a further approach to chondrocyteproliferation. This second approach has a focus upon adrenomedullin. Theapplicants have found that, in an equivalent manner to amylin,increasing the effective concentration of adrenomedullin within apatient able to interact with the chondrocytes in that patientstimulates chondrocyte proliferation.

For use in this approach, adrenomedullin can be obtained from anyconvenient commercial source or, as is the case with amylin, synthesisedusing techniques well known in the art. Such techniques include thosedescribed hereinafter.

Again, it is most convenient that the effective concentration ofadrenomedullin be increased through direct administration using eitheradrenomedullin itself or an adrenomedullin pro-drug. However,adrenomedullin agonists or inhibitors of adrenomedullin antagonists arenot excluded.

As with amylin, adrenomedullin can also be administered in the form of areplicable vehicle encoding adrenomedullin to the patient for release ofadrenomedullin over a prolonged period.

Adrenomedullin analogs can also be employed. For this purpose, the term“analog” has the equivalent meaning of that given above for amylin. Inthe case of adrenomedullin, a particularly preferred analog isadrenomedullin (27-52) (ie. a fragment consisting of amino acids 27-52of the adrenomedullin sequence).

The invention still further provides a third approach to chondrocyteproliferation. This further approach focuses upon the receptors onchondrocytes to which amylin and adrenomedullin bind and upon effectingchondrocyte proliferation through use of any ligand which both binds toand activates these receptors.

It will be appreciated that amylin analogs of amylin, adrenomedullin andanalogs of adrenomedullin are all ligands which achieve this. Indeed,the use of these substances as active agents represents a preferredaspect of the invention. However, it should be appreciated that thisapproach has not restricted the use or amylin, adrenomedullin and theiranalogs but also extends to any ligand which fulfils the functionalrequirement of both binding to and activating (stimulating) the amylinor adrenomedullin receptors. Such additional ligands are, for example,believed to include peptides such as calcitonin gene related peptide(Muff, R., et al. Calcitonin, calcitonin gene-related peptide,adrenomedullin and amylin: homologous peptides, separate receptors andoverlapping biological actions. Eur. J. Endocrinol 133:17-20 (1995)).

A specific feature of this approach is to employ ligands which bind toand activate the adrenomedullin receptor. This receptor was describedin, for example, Kapas, S., et al. Cloning and expression of cDNAencoding a rat adrenomedullin receptor. J. Biol. Chem. 270:25344-25347(1995). It is further described in Montuenga, L. M., et al. Expressionof adrenomedullin and its receptor during embryogenesis suggestsautocrine or paracrine modes of action. Endocrinology 138:440-451(1997)).

Additional stimulatory ligands can therefore, for example, be identifiedby a screening protocol employing at least the ligand binding domain ofthe adrenomedullin receptor. This screening method can, for example,utilise the expression of the adrenomedullin receptor in Xenopus oocytesusing standard recombinant DNA methods and measurement of theadrenomedullin receptor-mediated signal transduction evoked by novelstimulatory ligands.

For therapeutic application, the active compound (amylin, adrenomedullinanalog or ligand) will be formulated as a medicament. The details of theformulation will ultimately depend upon the active compound itself andupon the route of administration chosen. It will however be usual forthe medicament to include combination of the active compound with asuitable carrier, vehicle or diluent.

Dosage rates will also be active compound and administration routedependent. However, by way of example, the dosage of active compound tobe administered by injection will be in the range of 0.01-100 mg/kg ofbody weight

Further, while formulations in which the active compounds represent thesole active principle are most likely to be used, it is by no meansintended that formulations which are suitable for combination therapiesbe excluded. The active compound can be administered together with anyother therapeutic agent, including any other agent which has an effecton chondrocyte proliferation.

The invention, in its various aspects, will now be illustrated by theexperimental section which follows. It will however be appreciated thatthe experiments are non-limiting.

Experimental

Methods

(a) Chondrocyte Monolayer Cell Cultures

Fresh cartilage samples were collected from the tibial plateaus andfemoral condyles of mature, healthy crossbred dogs 12-4 years, 20-25).The chondrocytes were isolated as previously described (ConnectiveTissue Research 1988; 18:205-222). Briefly, the chondrocytes wereobtained by pronase and collagenase digestion of the cartilage, then thecells were centrifuged, washed and resuspended in media before beingcultured in 75 cm² tissue culture flasks. The cells were incubated under5% CO₂ and 95% air at 37° C. Confluence was reached by 7-10 days, atwhich time the cells were subcultured. After trypsinization, the cellsare rinsed and resuspended in fresh medium, then seeded at 5×10⁴cells/ml in 24-well plates (0.5 ml cell suspension per well, ie. 1.4×10⁴cells/cm²). Proliferation studies (cell counts and thymidineincorporation) were performed. Subconfluent population were changed toserum-free medium with 0.1% bovine serum albumin plus the experimentalcompounds. Cell numbers were analysed at 24 hours after addition of thepeptide or vehicle. The cell numbers were determined using ahaemocytometer chamber. Results were expressed per well. [³H]-thymidineincorporation was assessed by pulsing the cells with [³H]-thymidine(luCi/well) two hours before the end of the experimental incubation. Theexperiment was terminated at 24 hours by washing the cells in mediacontaining cold thymidine followed by the addition of 10%tricholoroacetic acid. The precipitate was washed twice with ethanolether (3:1) and the wells desiccated at room temperature. The residuewas redissolved in 2 M KOH at 55° C. for 30 mins, neutralised with 1 MHCl, and an aliquot counted for radioactivity. Results were expressed asdpm per well. For both cell counts and thymidine incorporation, eachexperiment was performed at least 4 different times using experimentalgroups consisting of at least 6 wells.

(b) Chondrocytes 3-Dimensional Cell Cultures In Alginate Beads Alginatehead cultures were established as described by Guo, et al. Culture andgrowth characteristics of chondrocytes encapsulated in alginated beads.Connective Tissue Research 19:277-297 (1989). Briefly the cells weresuspended in a solution of 1.25%/wt/vol) alginate in HEPES (20 mM HEPESbuffer pH neutral) at a density of 2×10⁶ cells/ml> The suspension ofchondrocytes were slowly extruded through a 22-gauge needle in adropwise manner into 40 ml of 0.1 M CaCl₂ solution. After instantaneousgelation, the beads were allowed to further polymerise in CaCl_(/2)solution (10 mins, room temperature). The beads were washedsequentially, twice in 0.15 M NaCl and twice in Dulbecco's modifiedEagle's medium (DME). After the washing procedure, the beads were placedinto 24-well culture plates (10 beads/well) and fed with 1 ml 10% fetalcalf serum (FCS) SMW with 5 μg/ml ascorbic acid. The cultures weremaintained at 37° C. in a humidified atmosphere of 5% CO₂ in air. Themedium was changed every second day. On day 4 and 6 of culture, peptideor vehicle was added. Cell numbers were analysed at day 8 by exposingalginate beads to 50 mM ethylenediaminetetraacetic acid (EDTA) forapproximately 10 minutes at 37° C. Counting was performed in ahaemocytometer chamber. Results were expressed per well.Tritiated-thymidine incorporation (³H-thymidine) was assessed by pulsingthe beads with ³H-thymidine (1 μCi/well) 48 hours before the end ofexperimental incubation. Experiments were then terminated at day 8 ofculture by dissolving the beads in 50 mM EDTA. The cells were washedtwice with distilled water by centrifuging. Pellets were resuspended andcounted for radioactivity.

(c) In Vivo Study: Experimental Design

Two groups of 20 sexually mature male Swiss mice aged between 40 and 50days and weighing 25-32 g, were given daily subcutaneous injections (50ul) in the loose skin at the nape of the neck for 5 days/week over 4consecutive weeks. The treated group was injected with peptide at a doseof 300 ug/kg/injection and the control group was injected with vehicle(water). Animals were housed in a room maintained at 20° C. on 12-hourlight/dark cycles. They were fed diet 86 rodent pellets (New ZealandStockfeed Ltd) ad libitum throughout the experiment. Each animal'sweight was recorded at the beginning and end of the experiment One dayafter the last injection, animals were sacrificed by cervicaldislocation. They study had the approval of the local institutionalreview board.

The tibiae were dissected free of adherent tissue. Tibial lengths wererecorded by measuring the distance between the proximal epiphysis andthe distal tibio-fibular junction using an electronic micrometerDigimatic Calipers, Mitutoyo, Japan). Bones were placed in 10%phosphate-buffered formalin for 24 hours and then dehydrated in a gradedseries of ethanol solutions and embedded, undecalcified, inmethylmethacrylate resin. Tibiae were sectioned longitudinally throughthe frontal plane and calvariae were cut cross-sectionally at the baseof the parietal bone. All sections were 4 um think and were cut on aLeitz microtome using a tungsten-carbide knife (Microknife Sharpening,Utah, USA). Sections were mounted on gelatin-coated slides andair-dried. They were stained with Goldner's tri-chrome and examinedusing an Olympus BX 50 microscope (Olympus Optical Co Ltd, Tokyo, Japan)which was attached to an Osteomeasure Image Analyzer (Osteometrics Inc.Atlanta, Ga.).

Tibial histomorphometric analyses were made from three adjacent sectionsone third of the way through the anterior/posterior depth of theproximal tibiae. Epiphyseal growth plate thickness was measured at threesites evenly spaced along its length. All measurements were made by oneoperator who was blinded to the treatment group of each bone.

Materials

Rat amylin was sourced from Bachem Calif., Torrance, Calif., USA.Lyophilised material was dissolved in water prior to administration.Methylmethacrylate was purchased from Acros Organics N.V., Geel,Belgium.

Rat amylin-(1-8) used in this study was a COOH-terminal amidesynthesized on methylbenzhydrylamine resin by standard solid-phasetechniques followed by hydrogen fluoride deprotection and cleavage fromthe resin. Amylin-(1-8) was cyclized in a dilute solution of 90% aceticacid by treatment with methanol solutions of iodine and punned to >96%homogeneity by reverse-phase high performance liquid chromatograph (RPHPLC). Structures were confirmed by matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF system, modelG2025 A, Hewlett Packard CA, USA) and amino acid analysis of acidhydrolysates %%4929%%.

Human adrenomedullin and its fragments were synthesized onmethylbenzhydxylamine resin using standard solid-phase procedures, andcleaved with hydrogen fluoride/anisole. Sequences containing a disulfidebridge were cyclized by titration with 1 ₂ in 90% acetic acid/watersolutions. Crude materials were purified by gel filtration on Sephadexcolumns in 50% acetic acid followed by gradient elution on C18 silicausing acetonitrile/0.1% trifluoroacetic acid eluants. Homogeneity offinal peptides was assessed by thin layer chromatography, analyticalHPLC, amino acid analysis and matrix-assistedlaser-desorption-ionization mass spectroscopy. Purities were usually>98%.

Statistical Analysis

Data are presented as mean±sem. Where parameters have been measured morethan once in each animal these values have been averaged to produce asingle value for each animal before further analysis. The significant oftreatment effects was evaluated using Student's t tests for unpaireddata. These comparisons were specified a priori, so adjustment ofα(0.05) was not necessary.

Results

Amylin

(a) Chondrocyte Cell Cultures

Amylin influenced chondrocyte proliferation, increasing cell numbersfrom 4.12±0.23 (×10⁴) (mean±sem) in control cells to 5.11±0.21 (×10⁴) inthose cells incubated with amylin (p=0.01) as well as increasingthymidine incorporation (ie. DNA synthesis) from 20725±997 dpm incontrol cells to 25937±1203 dpm in amylin-treated cells.

(b) Chondrocytes 3-Dimensional Cell Cultures In Alginate Beads

Amylin again influenced chondrocyte proliferation, increasing cellnumbers from 5.58±0.16 (×10⁴) (mean±sem) in control cells to 6.07±0.05(×10⁴) in those cells incubated with amylin (10⁻¹⁰M) (p<0.03) as well asincreasing thymidine incorporation (ie. DNA synthesis) from 1135±85 dpmin control cells to 2584=229 dpm in amylin-treated cells (p<0.01).

(c) In Vivo Study

Amylin influenced the tibial growth plate, increasing its width from0.083±0.005 mm (mean±sem) in the control animals to 0.108±0.003 mm inthose receiving amylin (P=0.0002) (FIG. 1). The total length of thetibiae was also increased from 11.31±0.07 mm in control animals to11.67±0.09 mm in animals injected with amylin (P-0.004) (FIG. 2).

Amylin 1-8

(a) Amylin-(1-8) also influenced chondrocyte proliferation, increasingcell numbers from 3.23±0.11 (×10⁴) (mean±sem) in control cells to3.63±0.09 (×10⁴) in those cells incubated with amylin-(1-8) (10-8M)(p=0.02) as well as increasing thymidine incorporation (DNA synthesis)from 26859±423 dpm in control cells to 28932±628 dpm in amylin-(1-8)treated cells (p=0.02).

(c) The growth plate width in the proximal tibiae of mice injectedsystemically with amylin-(1-8) is significantly increased compared tocontrol animals (mean±sem: 0.111 mm±0.004 compared to 0.081 mm±0.004;p<0.000 I). See FIG. 3.

Adrenomedullin

(a) Adrenomedullin influenced chondrocyte proliferation, increasing cellnumbers from 1.79±0.07 (×10⁴) (mean±sem) in control cells to 2.27±0.12(×10⁴) in those cells incubated with adenomedullin (10 ⁻⁹M) (p<0.01).

Adrenomedullin-(27-52)

(c) Adrenomedullin-(27-52) increased the growth plate width from 0.094mm±0.003 (mean±sem) in control animals to 0.11 mm±0.003 inadrenomedullin-(27-52) (p=0.003). See FIG. 4.

Industrial Application

The above results clearly show that amylin and its anlogs (amylin-(1-8)for example) has the ability to stimulate chondrocyte proliferation.Similarly, adrenomedullin and its analogs (adrenomedullin-(27-52) haveequivalent capability.

The results additionally show the ability of both amylin, adrenomedullinand their analogs to influence the growth of cartilage as well asincreased bone growth. This latter effect is consistent with theformation of bone on a template of cartilage tissue.

Both amylin and adrenomedullin are believed to be exerting the effect onchondrocyte proliferation/cartilage growth/bone formation through themediation of the amylin/adrenomedullin receptor.

The present invention therefore provides new approaches to chondrocyteproliferation. These involve firstly increasing the active concentrationof amylin/adrenomedullin in a patient and secondly the activation of theamylin/adrenomedullin receptor localised on chondrocyte cells.

The approaches of the invention have application in the treatment ofpatients in a variety of conditions. Principal amongst these areconditions where the patient is suffering from a cartilage defect,either through injury or through degenerative, inflammatory or otherdisease.

The approaches of the invention also have application in the stimulationof bone growth, particularly lineal bone growth. This provides theinvention with application in treating patients (for example, children)who are of short stature or who otherwise suffer from defects whichwould benefit from stimulation of the growth of limb bones.

The invention also has application in vitro. Extracted chondrocytes canbe proliferated using the present methods. The proliferated chondrocytescan then be employed in methods of therapy, particularly those whichinvolve the treatment of damaged cartilage.

It will be appreciated by those persons skilled in the art that theabove description is provided by way of example only and that numerouschanges and variations can be made while still being within the scope ofthe invention as defined by the appended claims.

1. A method of treating a patient to stimulate chondrocyte proliferationin vivo which comprises the step of increasing amylin activity withinsaid patient by an amount effective to stimulate chondrocyteproliferation.
 2. A method of treating a patient to stimulate cartilagegrowth or repair in vivo through stimulation of chondrocyteproliferation which comprises the step of increasing amylin activitywithin said patient by an amount effective to stimulate chondrocyteproliferation.
 3. A method of treating a patient to stimulatechondrocyte proliferation in vivo which comprises the step ofadministering to said patient amylin or an analog thereof in an amounteffective to stimulate chondrocyte proliferation, the amylin analogbeing a fragment of amylin comprising amylin (1-8) (SEQ ID NO:2).
 4. Amethod of treating a patient to stimulate cartilage growth or repair invivo through stimulation of chondrocyte proliferation which comprisesthe step of administering to said patient amylin or an analog thereof inan amount effective to stimulate chondrocyte proliferation, the amylinanalog being, a fragment of amylin comprising amylin (1-8) (SEQ IDNO:2).
 5. A method of stimulating chondrocyte proliferation in vitrowhich comprises administering an amount of amylin or an analog of amylinto said chondrocytes is in an amount effective in inducing chondrocyteproliferation, the amylin being a fragment of amylin comprising amylin(1-8).